1. Brief Discussion of the Related Art
Since ancient times, people keep seeking for the way or panacea to preserve youth and immortality. In recent years, a fashion of anti-aging slowly spread to the global with the successive development and marketing of anti-aging products. In Japan, for instance, more than 70% people become aware of anti-aging. People in Taiwan also gradually increase the concern for anti-aging, which will promote the marketing of anti-aging products, and is expected to continue expanding in the global market.
Taking the case of anti-aging products marketing in Taiwan as an example, market scale of which has been grown by 17 times within the last decade, with increased from the sum of business of 20 billion NT dollars in 2001 to 25.7 billion NT dollars in 2002, and to 41.2 billion NT dollars in 2006. The estimated business in the next 10 years will be raised to 400 billion NT dollars. This shows that consuming power in the market of anti-aging beauty and health care products is really amazing, and is not influenced by the economic cycle. Therefore, it is in need of developing a high-throughput assay for rapid and efficient screening anti-aging agents in known artificial drugs or natural products.
When human cells are placed in a culture environment, they exhibit a finite proliferative capacity and are usually able to divide only forty to sixty times before reaching a senescent (nondividing) phase. The limited proliferative capacity of human cells in a culture environment is thought to result from multiple environmental and genetic mechanisms, and has been widely used as a model of human aging.
Cellular senescence was observed and proposed as a model for aging at the cellular level over thirty years ago. Leonard Hayflick studied human fibroblast like cells obtained from lung and skin. Hayflick found that when serially cultured, these cells would undergo rounds of divisions, but as the culture aged, the cells were no longer able to divide. In conjunction with the loss of division potential, there were changes in the morphology, the shape and physical appearance, of cells. The cells enlarged significantly, and more space was observed between individual cells.
In prior art, senescence-associated β-galactosidase (SA-β-gal) assay has been used in determination of cellular senescence in culture. The assay is based on finding that senescence induces the increase in levels of lysosomal β-galactosidase. In nonsenescent cells, the lysosomal hydrolase β-galactosidase cleaves galactose from glycoproteins at an optimum pH of 4.0 to 4.5. Lysosomal β-galactosidase activity can be detected in most mammalian cells by performing a cytochemical assay at pH 4.0 in which cleavage of X-gal by β-galactosidase leads to the formation of a blue precipitate. However, during senescence, there is an increase in lysosomal β-galactosidase protein levels and an overall increase in lysosomal size. The increase in β-galactosidase levels allows the detection of β-galactosidase activity at the suboptimal pH of 6.0 during senescence (Kurz et al., 2000, J Cell Sci. 113: 3613-3622).
Telomere and telomerase assay is an alternative method used in determining cell aging. At the end of a chromosome is a telomere, which keeps chromosome protected and prevent it from fusing into rings or binding with other DNA. When a cell divides and copies DNA, the telomeres get snipped to enable the copying process. Researchers can use the length of a cell's telomeres to determine the cell's age and how many more times is will replicate. In 1985, scientists discovered telomerase, a reverse transcriptase composed of two primary components, the protein catalytic subunit, TERT, and the template RNA subunit, TR. This enzyme extends telomeres, rebuilding them to their former lengths, by using its own RNA as a template for adding nucleotides to the ends of chromosomes. Detection of the expression of human catalytic subunit of telomerase (hTERT) provides an assay for analysis of telomerase activity on its native substrate, telomeres.
2. Field of the Invention
In present invention, an analytic method of cell aging is provided by detecting the expression level of cofilin in target cells. Cofilin is a ˜19 kD protein that can bind to actin filaments and promote their dynamics for motility, development, polarity or cytokinesis (Bamburg et al., 1999, Trends Cell Biol 9: 364-70). Three isoforms of cofilin, including nonmuscle cofilin (n-cofilin), muscle cofilin (m-cofilin) and ADF, are expressed in mammals. Loss of cofilin expression leads to G2/M phase arrest, the formation of multinucleate cells and irreversible cell death (Bellenchi et al., 2007, Genes Dev 21: 2347-57). However, there are no related references ever disclosed the correlations of endogenous cofilin expression level and the cellular aging situation in target cells.